Injection Of Estradiol Research Articles

8309 Injectable Estradiol Dosing Regimens; A Retrospective Review of Hormone Therapy for Gender-Diverse Adults Assigned Male at Birth

Abstract Disclosure: M. Kanin: None. M. Slack: None. R. Patel: None. K. Chen: None. N. Jackson: None. K. Williams: None. S. Grock: None. Introduction: Many gender incongruent individuals assigned male at birth (AMAB) seek hormone therapy to achieve physical and emotional changes. Hormone therapy typically includes estradiol, with or without an anti-androgen. However, limited data with relatively weak justifications support the currently recommended regimens. In particular, dosing schedules for injectable estradiol can vary considerably. UCSF guidelines recommend initiating estradiol valerate at 5 mg parenterally weekly or estradiol cypionate 2 mg weekly. Endocrine Society Guidelines recommend estradiol valerate or cypionate parenterally 5-30 mg every 2 weeks or 2-10 mg weekly. Our clinical observations led to the concern that current guideline-based dosing of injectable estradiol can result in supratherapeutic estradiol levels. Thus, we designed a retrospective cohort study to evaluate injectable estradiol dosing requirements for gender diverse individuals at our institution. Hypothesis: We hypothesize that low dose estradiol injections can lead to therapeutic estradiol levels (100-200 pg/mL) and suppressed testosterone (<50 ng/dL). Design: We performed a retrospective cohort study of gender diverse individuals AMAB aged 18-89 who were newly prescribed injectable estradiol by a single clinic from January 2017 to March 2023. Exclusion criteria included history of orchiectomy or estradiol use within the previous six months. Patients were identified using structured query language to extract data from our institution’s electronic health record system. A total 33 patients were identified, 29 of whom were eligible for inclusion. Data was collected for 15 months after estradiol initiation. Results: All individuals were prescribed estradiol valerate except for 1 who was prescribed estradiol cypionate. Estradiol dose ranged from 2.5 to 10 mg weekly and an average initial dose of 4.26 mg weekly. Mean initial estradiol level obtained while on treatment for at least 30 days was 349.8 pg/mL, which is supratherapeutic. Mean initial testosterone level was 25.3 ng/dL, which is suppressed. Mean estradiol dose at completion of data collection was 3.68 mg with a mean final estradiol level of 245.2 pg/mL. Testosterone levels reached less than 50 ng/dL for everyone in the sample with a mean final testosterone of 23.3ng/dL despite significant reductions in estradiol dose (change=-0.58 mg per week, p=0.001) Conclusion: Our study demonstrates lower dosages of estradiol can be used to achieve therapeutic estradiol levels in gender incongruent patients seeking estrogen therapy. Using lower dosages can avoid supratherapeutic estradiol levels and the associated risks. Further prospective studies are needed with the aim to reevaluate current guideline dosing recommendations for injectable estradiol. Presentation: 6/2/2024

8309 Injectable Estradiol Dosing Regimens; A Retrospective Review of Hormone Therapy for Gender-Diverse Adults Assigned Male at Birth

Abstract Disclosure: M. Kanin: None. M. Slack: None. R. Patel: None. K. Chen: None. N. Jackson: None. K. Williams: None. S. Grock: None. Introduction: Many gender incongruent individuals assigned male at birth (AMAB) seek hormone therapy to achieve physical and emotional changes. Hormone therapy typically includes estradiol, with or without an anti-androgen. However, limited data with relatively weak justifications support the currently recommended regimens. In particular, dosing schedules for injectable estradiol can vary considerably. UCSF guidelines recommend initiating estradiol valerate at 5 mg parenterally weekly or estradiol cypionate 2 mg weekly. Endocrine Society Guidelines recommend estradiol valerate or cypionate parenterally 5-30 mg every 2 weeks or 2-10 mg weekly. Our clinical observations led to the concern that current guideline-based dosing of injectable estradiol can result in supratherapeutic estradiol levels. Thus, we designed a retrospective cohort study to evaluate injectable estradiol dosing requirements for gender diverse individuals at our institution. Hypothesis: We hypothesize that low dose estradiol injections can lead to therapeutic estradiol levels (100-200 pg/mL) and suppressed testosterone (<50 ng/dL). Design: We performed a retrospective cohort study of gender diverse individuals AMAB aged 18-89 who were newly prescribed injectable estradiol by a single clinic from January 2017 to March 2023. Exclusion criteria included history of orchiectomy or estradiol use within the previous six months. Patients were identified using structured query language to extract data from our institution’s electronic health record system. A total 33 patients were identified, 29 of whom were eligible for inclusion. Data was collected for 15 months after estradiol initiation. Results: All individuals were prescribed estradiol valerate except for 1 who was prescribed estradiol cypionate. Estradiol dose ranged from 2.5 to 10 mg weekly and an average initial dose of 4.26 mg weekly. Mean initial estradiol level obtained while on treatment for at least 30 days was 349.8 pg/mL, which is supratherapeutic. Mean initial testosterone level was 25.3 ng/dL, which is suppressed. Mean estradiol dose at completion of data collection was 3.68 mg with a mean final estradiol level of 245.2 pg/mL. Testosterone levels reached less than 50 ng/dL for everyone in the sample with a mean final testosterone of 23.3ng/dL despite significant reductions in estradiol dose (change=-0.58 mg per week, p=0.001) Conclusion: Our study demonstrates lower dosages of estradiol can be used to achieve therapeutic estradiol levels in gender incongruent patients seeking estrogen therapy. Using lower dosages can avoid supratherapeutic estradiol levels and the associated risks. Further prospective studies are needed with the aim to reevaluate current guideline dosing recommendations for injectable estradiol. Presentation: 6/2/2024

Pregnancy history and estradiol influence spatial memory, hippocampal plasticity, and inflammation in middle-aged rats

Pregnancy and motherhood can have long-term effects on cognition and brain aging in both humans and rodents. Estrogens are related to cognitive function and neuroplasticity. Estrogens can improve cognition in postmenopausal women, but the evidence is mixed, partly due to differences in age of initiation, type of menopause, dose, formulation and route of administration. Additionally, past pregnancy influences brain aging and cognition as a younger age of first pregnancy in humans is associated with poorer aging outcomes. However, few animal studies have examined specific features of pregnancy history or the possible mechanisms underlying these changes. We examined whether maternal age at first pregnancy and estradiol differentially affected hippocampal neuroplasticity, inflammation, spatial reference cognition, and immediate early gene activation in response to spatial memory retrieval in middle-age. Thirteen-month-old rats (who were nulliparous (never mothered) or previously primiparous (had a litter) at three or seven months) received daily injections of estradiol (or vehicle) for sixteen days and were tested on the Morris Water Maze. An older age of first pregnancy was associated with impaired spatial memory but improved performance on reversal training, and increased number of new neurons in the ventral hippocampus. Estradiol decreased activation of new neurons in the dorsal hippocampus, regardless of parity history. Estradiol also decreased the production of anti-inflammatory cytokines based on age of first pregnancy. This work suggests that estradiol affects neuroplasticity and neuroinflammation in middle age, and that age of first pregnancy can have long lasting effects on hippocampus structure and function.

Genetically engineered mouse model of HPV16 E6-E7 with vaginal-cervical intraepithelial neoplasia and decreased immunity

ObjectiveTo construct models of high-risk human papillomavirus (HPV) infection with precancerous lesions or cervical cancer and explore the immune function. MethodsUsing CRISPR/Cas9, the expression vector HPV16-E6-E7-Rosa26 was microinjected into fertilized eggs of C57BL/6 N mice using homologous recombination, and the F0 generation was obtained for reproduction. Then, the formation of precancerous lesions was promoted via intramuscular injection of estradiol. Presence of precancerous cervical-vaginal intraepithelial lesions, Ki67 and p16 expression levels, and CD8+ T cell proportions in the spleen were evaluated. ResultsTwo F0 generation mice exhibited correct the homologous recombination. Seven positive mice were identified in the F1 generation. After breeding and mating, 25 homozygous and 11 heterozygous HPV16-E6-E7-engineered mice were obtained from the F2 generation. After estradiol benzoate treatment, the cervical-vaginal epithelium appeared as precancerous lesions with positive Ki67 and p16 expression. The percentage of CD8+ T cells decreased. ConclusionHPV16-E6-E7-Rosa26 induced low immune function in mice, and provides a good model for the basic research of the mechanisms of action of HPV infection-associated precancerous lesions or cervical cancer.

Effects of estradiol on fatty acid composition and tissue structure of hepatopancreas in Procambarus clarkii

The hepatopancreas of Procambarus clarkii provides lipids and other nutrients for ovarian development and contains hormones such as estradiol related to ovarian development. During the development of the P. clarkii ovary, the hepatopancreas’ tissue structure and fatty acid content undergo corresponding changes. A controlled indoor experiment was conducted to investigate the effects of exogenous estradiol injection on the tissue structure and fatty acid content in the hepatopancreas of P. clarkii. The experiment consists of three experimental groups and one control group. The P. clarkii in the experimental group were injected with 5, 0.5, and 0.05 μg/(g body weight) of estradiol once every 5 days for 5 times and with normal saline injection as the control. After 25 days, the content of fatty acids and changes in tissue structure in the hepatopancreas were measured. The results showed that the main fatty acids in P. clarkii hepatopancreas were C18:4n, C20:3n, and EPA, and polyunsaturated fatty acid (PUFA) content was 57.17%±4.76%. Injecting estradiol mainly affected the content of C16:0, C16:1n, C18:0, C18:1n9, C18:2n, C18:3n6, C18:3n6, C18:4n, C20:1n, C20:2n, C20:3n, C20:4n3, C20:5n3, C22:n (n=1-4), C22:5n, and C22:6n in the hepatopancreas. Injecting estradiol significantly reduced the total content of PUFA in hepatopancreas with the reduction of C18:3n6, C18:4n, C20:3n, C20:4n3, C20:5n3 C22: n (n=1-4) (P<0.05); Estradiol injection significantly increased the total content of monounsaturated fatty acid (MUFA) in hepatopancreas with the increase of C18:1n9, and C20:1n (P<0.05); Injection of estradiol (0.05 μg/g) significantly increased the total content of saturated fatty acids (SFA) in the hepatopancreas (P<0.05) with the increase of C16:0 and C18:0 (P<0.05). Injecting estradiol can increase the volume and quantity of hepatopancreatic B cells, and the 5 μg/g group had the most obvious changes.

Changes in the femoral trochlear induced with estradiol exposure, an experimental study on rats.

It is well known that estrogen is closely related to bone and joint tissue. Findings indicate that estradiol, injected during pregnancy, passes through the placental barrier and reaches the fetuses in utero where it exerts its action. Since trochlea appears well established in the prenatal period, however, whether trochlear dysplasia is related to estradiol exposure has not been confirmed, and the pathological process of estradiol exposure-induced trochlear dysplasia remains unclear. This study aimed to establish an estradiol exposure animal model in fetuses and to analyze the morphology of the femoral trochlear in neonatal rats. 30 pregnant Wistar rats provided by the local Animal Center were assigned randomly into three groups, a high dose estradiol injection group, a low dose estradiol injection group and a blank control group. Gross, cross-sectional observation, histological staining measurement and microcomputed tomography of the rat offspring were conducted to evaluate the morphological changes of the femoral trochlea. The incidence of trochlear dysplasia increased with the concentration of estradiol injection. Gross and cross-sectional observation showed a shallower trochlea groove in two groups with estradiol injection. Histological staining measurement indicated that the trochlear sulcus angle and trochlear sulcus depth were significantly different between the two groups with estradiol injection and the blank control group at 0,5 and 10 days after birth. Subchondral bone loss was observed in the two estradiol injection groups by micro-CT, and the bone loss was found to deteriorate over time. In this study, estradiol exposure in fetuses had an adverse effect on intrauterine development and could induce trochlear dysplasia and bone loss in rat offspring. In addition, this study also showed that the higher concentration of estradiol injection in pregnant rats, the more incidence of trochlear dysplasia in rat offspring.

Electroacupuncture stimulating Zhongji (CV3), Guanyuan (CV4), and bilateral Dahe (KI12) attenuates inflammation in rats with chronic nonbacterial prostatitis induced by estradiol through inhibiting toll-like receptor 4 pathway.

To investigate the anti-inflammatory effect of electroacupuncture (EA) on chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), also known as chronic nonbacterial prostatitis (CNP), and explore its underlying mechanism. A CNP rat was established by surgical castration combined with 17-β estradiol injection in male Sprague-Dawley rats for thirty consecutive days. The CNP rats received EA treatment once a day for eight days. Chronic pelvic pain was evaluated by mechanical withdrawal threshold measurement. The histological change was assessed by hematoxylin-eosin staining. The inflammatory cytokines in prostates were determined by enzyme-linked immunosorbent assays. The expressions of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), inhibitors of kappa-B alpha (IκBα), and nuclear factor-kappa B (NF-κB) were detected by Western blotting. The nuclear translocation of NF-κB and the location of TLR4 were observed with immunofluorescence staining. The results showed that EA decreased the prostate index, upregulated the mechanical withdrawal threshold, restored the histomorphology of the prostate, reduced the inflammatory factor levels, inhibited NF-κB p65 nuclear translocation, and downregulated the expression levels of critical proteins involved in the TLR4/NF-κB signaling pathway in prostates. Our findings suggested that EA could relieve pelvic pain and attenuate prostatic inflammation in estradiol-induced CNP rats. The underlying mechanism may be related to the inhibition of the TLR4/NF-κB signaling pathway.

Ovarian activity and reproductive responses of lactating Angus cows due to a mineral supplementation throughout a timed AI protocol.

Two experiments were conducted to evaluate the effect of intramuscular administration of minerals during a TAI program on the reproductive responses of lactating Angus cows. All cows (n=353) were subjected to a 9-day TAI program based on CIDR insertion plus injections of estradiol, cloprostenol, and eCG, and then TAI 48 h later. In experiment 1, two groups were randomly created, one control with a placebo injection (CON, n=109), and the second received 10 mL of Fosfosan® (MIN, n=172) on day 0 of the synchronization. Conception rate (66.9 vs. 55%) and estrus percentage (55.8 vs. 44%) were higher (P≤0.05) in MIN than in CON cows. Given these results, a second experiment was conducted randomly assigning the cows to two treatments (n=36 each): a single injection of 10 mL of Fosfosan® (MIN-O) on day 0 or two injections of 10 mL of Fosfosan® (MIN-T) on synchronization days 0 and 7. Four cows of each treatment were randomly selected to be scanned with transrectal ultrasound before and during the synchronization protocol to assess ovarian structures and cyclicity, and at day 39 post-TAI for pregnancy diagnosis. Also, blood samples were obtained for the determination of serum minerals and progesterone (P4) concentrations. The number of mineral injections did not affect conception rate (P≥0.1229) conception rate, serum mineral and P4 concentrations, number, and size of emerging follicles, or follicle size according to 1 to 4 classifications. The MIN-T promoted (P<0.05) earlier follicular wave emergence than MIN-O. However, MIN-O cows had a dominant follicle of 15.12 mm, which is more significant (P<0.05) than that in MIN-T cows (13.5 mm). In conclusion, providing a single mineral injection of Fosfosan® at the start of a TAI program is an excellent reproductive strategy in lactating Angus cows to improve the dominant follicle growth, estrus response, and conception rate.

(017) Systemic Estradiol Menopause Management: Return to the Old-Fashioned Way

Abstract Introduction The mean systemic estradiol value in menopausal women is &amp;lt; 10 pg/ml, consistent with ovarian failure. Such low values are associated with multiple symptoms including hot flashes, night sweats, insomnia, mood changes and irritability, cognitive changes, weight gain and slowed metabolism. Contemporary management with systemic estradiol, maximizing safety and efficacy, emphasizes non-oral, non-injection estradiol replacement therapies including systemic daily gels, twice weekly or weekly patches or three-month vaginal rings. Most women (&amp;gt;60%) who try these systemic therapies in our sexual medicine clinic have been successful at reducing bothersome symptoms. We have identified a subset of women who cannot achieve sufficient symptom relief despite using contemporary estradiol strategies due to lack of efficacy, negative side effects, (rings cannot stay in the vagina or values are too high initially and then too low, patches cause rashes or fall off, gels are too messy) or financial concerns. For these patients, we have advised a return to the “old-fashioned way” using weekly intramuscular (IM) injection of either estradiol valerate or cypionate. Estradiol cypionate was first described as well as introduced for medical use in 1952. Objectives This study examines the experiences of this subset of women in one practice using weekly injections of estradiol valerate or cypionate for either efficacy or financial reasons. Methods This chart review identified a subset of 16 patients (mean age 59 +/− 8 years) at a single site who had been prescribed estradiol valerate or cypionate when symptoms of genitourinary syndrome of menopause (GSM) persisted despite use of estradiol replacement gels, patches or rings, or cost was prohibitive. We have advised weekly IM injection of either estradiol valerate (20 mg/ml; starting dose 0.05 ml or 1 mg) or estradiol cypionate (5 mg/ml; starting dose 0.2 ml or 1 mg). We compared relief of bothersome symptoms from earlier estradiol treatments to current IM injection and compared monthly costs of each treatment based on GoodRx prices, assuming medications were not covered by insurance. Results All patients using weekly injections of estradiol valerate or cypionate had their estradiol-based GSM symptoms ameliorated without complaint of inconvenience or discomfort using injections. The risks of IM injections of estradiol are similar to those of topical administration, requiring concomitant progesterone use in women with a uterus. Concerning finances, using a GoodRx coupon, a 5 ml bottle of estradiol valerate costs approximately $50 and should last at least one year, reducing monthly cost to approximately $5/month. A 5 ml bottle of estradiol cypionate costs approximately $150 and should last at least 6 months, thus about $25/month. In contrast, generic patches are approximately $40/month, gels $150/month and the ring $170/month. While there remains an abhorrence for injections, the motivation for systemic estradiol replacement superseded any inconvenience of using an injectable medication and the 27-gauge ½ inch needle is associated with minimal pain. No significant side effects were noted with IM injection. Conclusions For certain individuals, sometimes the “old-fashioned way” works better both in terms of efficacy and cost, providing relief of bothersome menopausal symptoms. Disclosure No.

PROTECTIVE POTENTIAL OF 17-β-ESTRADIOL ON OXIDATIVE STRESS AND RENAL METABOLISM IN AGED FEMALE RATS

Abstract Background and Aims The objective of this study was to observe the changes in activity of antioxidant enzymes, hepatic glucose homeostasis, lipogenic enzymes and lipid metabolism, serum lipid profile and liver function occurring in livers of female rats of 3, 12 and 24 months age groups, and to see whether these changes are restored to 3 months control levels rats after exogenous administration of steroid hormone estrogens (17-β-estradiol, E2). Methods The aged rats (12 and 24 months old) (n= 8 for each group) were given subcutaneous injection of 17beta estradiol (0.1 ug/g body weight) daily for one month. After 30 days of hormone treatment, experimental animals of all the groups were sacrificed and livers were isolated for further study. A detailed study was carried on non-enzymatic glutathione (GSH) and enzymatic antioxidants [superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT)], hepatic glucose homeostasis, lipogenic enzymes, lipid metabolism, serum aspartate aminotransferase (GOT), alanine aminotransferase (GPT),alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), phosphatase alkalin (PAL) as well as bilirubin level. Results The results obtained in the present work revealed that normal aging was associated with significant decrease in the activities of antioxidant enzymes, serum expression and an increase in hepatic glucose homeostasis, lipogenic enzymes and lipid profile and GGT, PAL, GOT, GPT, ALP as well as bilirubin level increased significantly in livers of aging female rats. Our data showed that exogenous administration of E2 brought these changes to near normalcy in aging female rats. Conclusions The present study showed that E2 treatment reversed the changes to normal levels. E2 treatment may be beneficial in preventing some of the age related changes in the liver by increasing antioxidant defences and decrease oxidative stress. E2 plays important role in the progression of chronic hepatic diseases.

Factors affecting pregnancy rates in acyclic recipient mares prepared with an intravaginal progesterone device

In acyclic embryo recipient mares, artificial cycle protocols are conventionally based on estradiol followed by long acting (LA) progesterone injections. The use of intravaginal devices has been encouraged because it allows a rapid resynchronization of recipients (Rigoleto-Júnior et al., Journal of Equine Veterinary Science 2021; 97:103325) and days of estradiol injections have also been discussed (Silva et al., Journal of Equine Veterinary Science 2021; 103:103669), but further studies are needed to develop more appropriate protocols. The present work aimed to evaluate factors that affect fertility in a commercial ET program. A total of 114 healthy mares (5-15yr-old) were used as embryo recipients and divided into two groups according to ovarian activity: cyclic (n=57) and acyclic (n=57, anestrus or transitional). Day-0 was defined as the day of ovulation in the cyclic group and the day of a 1g intravaginal P4 device (iP4) insertion in the acyclic group. Before iP4 insertion, 17β-estradiol doses varied from 0 to 10mg daily depending on the presence of natural or induced uterine edema that ranged from 3 to 7 days in duration. A one-by-one Pearson's Chi-square test was used to compare pregnancy rates between groups and the effects on pregnancy rate of 1) number of days of 17β-estradiol injections, 2) uterine edema (0-4 score)on D0 in the acyclic group, and 3) day of ET in both groups. Plasma P4 concentrations were evaluated daily by RIA in acyclic (n=10) and cyclic (n=13) mares from D0 to D7. Repeated measure models followed by Tukey's test were used to compare mean plasma P4 within groups. Pregnancy rates did not differ between acyclic (82%, 47/57) and cyclic (75%, 43/57) mares (P=0.5). Uterine edema on Day 0 (P=0.7) and day of ET (P=0.05) did not affect pregnancy rates of acyclic mares. Three days of estradiol treatment in acyclic mares resulted in a lower pregnancy rate (P=0.0425) than four days of injections. For cyclic recipient mares, day of ET affected pregnancy rates (P=0.0001) where D4 (96%, 26/27) showed higher pregnancy rates than D5 (66.7%, 12/18) and D6-7 (41.7%, 5/12), but D5 and D6-7 did not differ. Mean plasma P4 concentrations varied over days for both groups (P>0.05). In cyclic mares, mean plasma P4 increased daily to D5 (plasma P4 = 10ng/mL) followed by a plateau, whereas in the acyclic group, iP4 insertion caused an abrupt rise in plasma P4 concentration within 24h (plasma P4=5.67mg/mL) followed by a gradual daily fall to D7 (plasma P4=2.78mg/dL). In conclusion, the use of acyclic recipient mares prepared with iP4 results in similar pregnancy rates as in cyclic mares despite differences in the plasma P4 profile.

Cellular proliferation and apoptosis in Staphylococcus aureus–infected heifer mammary glands experiencing rapid mammary gland growth

Intramammary infections in nonlactating mammary glands are common and can occur during periods of rapid mammary epithelial cell (MEC) accumulation, which may ultimately reduce total MEC numbers. Reduced MEC numbers, resulting from impaired MEC proliferation and increased cellular apoptosis, are expected to reduce future milk yields. The objective of this study was to measure the degree of cellular proliferation and apoptosis in the epithelial and stromal compartment of uninfected and Staphylococcus aureus-infected mammary glands hormonally induced to grow rapidly. Nonpregnant heifers (n = 8) between 11 and 14 mo of age were administered supraphysiological injections of estradiol and progesterone for 14 d. One mammary gland of each heifer was randomly selected and infused with Staph. aureus (CHALL) while another mammary gland was designated as an uninfected control on d 8 of injections. Mammary tissues were collected on the last day of hormonal injections from center and edge parenchymal regions and subject to proliferation assessment via Ki-67 staining and apoptotic assessment via terminal deoxynucleotidyl transferase dUTP nick-end labeling. Differences in cellular proliferation between CHALL and uninfected control quarters were not apparent, but proliferation of MEC was marginally greater in edge parenchyma than in center parenchyma. Coincidently, CHALL quarters experienced a greater percentage of apoptotic MEC and lower percentage of stromal cells undergoing apoptosis than uninfected control quarters. This study also provides the first insight into the mechanisms that allow the mammary fat pad to be replaced by expanding mammary epithelium as edge parenchyma contained a greater percentage of apoptotic stromal cells than center parenchyma. When taken together, these data suggest that Staph. aureus intramammary infection impairs mammary epithelial growth through reductions in MEC number and by preventing its expansion into the mammary fat pad. These factors during periods of rapid mammary growth are expected to impair first lactation milk yield.

Effect of electroacupuncture on the morphology of prostate and degranulation of mast cells in rats with chronic nonbacterial prostatitis

To observe the effect of electroacupuncture (EA) on the morphology of prostate and degranulation of mast cells in prostate of rats with chronic nonbacterial prostatitis (CNP). Male SD rats were randomly divided into sham operation group, model group and EA group, with 8 rats in each group. CNP model was established by surgical castration combined with 17-β estradiol injection once daily for 30 days. EA was applied to "Zhongji" (CV3), "Guanyuan" (CV4) and bilateral "Dahe" (KI12) for 20 min, once daily for 8 days. The mechanical pain threshold of scrotum skin area was tested before modeling, after modeling and after intervention. The pathological morphology of the prostate was observed by HE staining. Collagenous fiber was observed by Masson staining. The infiltration of mast cells was observed by toluidine blue staining. The contents of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in prostate were determined by ELISA. The protein expression levels of tryptase and transforming growth factor β1 (TGF-β1) in prostate were detected by Western blot. A normal appearance with little inflammatory cell infiltration was observed in the prostate of the sham operation group. Gland atrophy, epithelial destruction, hyperemia and edema, diffuse inflammatory cell infiltration and visible collagen fiber deposition were observed in prostate of the model group. The degree of infiltration of inflammatory cells and collagen fiber deposition were reduced in the EA group. Compared with the sham operation group, mechanical pain threshold was decreased (P<0.01), while the collagen volu-me fraction (CVF) of prostate, the degranulated rate of mast cells, the protein expression levels of tryptase and TGF-β1, and the contents of IL-6 and TNF-α were increased (P<0.01) in the model group. Following EA intervention, compared with the model group, the mechanical pain threshold was increased (P<0.01), CVF of the prostate, the degranulated rate of mast cells, the protein expression levels of tryptase and TGF-β1, and the contents of IL-6 and TNF-α were decreased (P<0.05, P<0.01) in the EA group. EA can relieve pain and reduce inflammation and fibrosis of prostate in CNP rats, which may be related to inhibiting the degranulation of prostate mast cells and down-regulating the expression of inflammatory factors and TGF-β1.

Histological tissue structure alterations resulting from Staphylococcus aureus intramammary infection in heifer mammary glands hormonally induced to rapidly grow and develop

Intramammary infections (IMI) are common in nonlactating dairy cattle and are expected to impair mammary growth and development and reduce future milk production. The objective of this study was to histologically evaluate how IMI alter tissue structure in growing and developing heifer mammary glands. A total of 18 nonpregnant, nonlactating heifers between 11 and 14 mo of age were used in the present study. Heifers received daily supraphysiological injections of estradiol and progesterone for 14 d to stimulate rapid mammary growth and development. One-quarter of each heifer was subsequently infused with Staphylococcus aureus (CHALL) while a second quarter served as an uninfected control (UNINF). Heifers were randomly selected and euthanized either the last day of hormonal injections to observe IMI effects on mammary gland growth (GRO), or 13 d post-injections, to observe IMI effects on mammary development (DEV). Mammary tissues were collected from the center and edge parenchymal regions of each mammary gland for morphometric tissue area evaluation. For GRO tissues, CHALL quarters had less epithelial tissue area and marginally more intralobular stroma tissue area than UNINF quarters. Tissue areas occupied by luminal space, extralobular stroma, adipose, and lobular tissue were similar. For DEV tissues, area occupied by epithelium, luminal space, intralobular stroma, and extralobular stroma did not differ between quarter treatments, but UNINF quarters had more adipose tissue area and marginally less lobular area than CHALL quarters. Results indicate that IMI in growing and developing mammary glands reduces mammary epithelial growth and alters mammary gland development by impairing epithelial branching into the mammary fat pad. Taken together, these tissue changes before calving may have adverse effects on milk production. Therefore, an important focus should be placed on improving udder health in replacement heifers through management strategies that mitigate the deleterious effects of IMI and promote the positive development of the mammary gland.

The effect of estrogen and progesterone on melanin-concentrating hormone producing-neurons in brain areas related to reproductive behavior in lactating dams

The hypothalamus plays a role in reproductive cycle control, and it is a site of action of steroid hormones. Throughout the production of melanin-concentrating hormone (MCH), the hypothalamus shows adaptive changes during lactation. Therefore, in this work, we aimed to test the effects of estrogen and progesterone manipulation on MCH-immunoreactive (ir) neurons in hypothalamic brain areas related to reproductive behavior and on the MCH serum concentration. Our results show that the removal of steroid hormones by ovariectomy increases the number of MCH-ir neurons in the medial preoptic area (MPOA) and incerto-hypothalamic area (IHy) but not in the anterior part of the paraventricular nucleus of the hypothalamus (PVHa). The MCH in the serum levels also increases. In accordance, the injection of estradiol alone or estradiol and progesterone decreased the number of MCH-ir neurons in the MPOA and IHy, as well as its serum levels. The MPOA and IHy are the brain areas targeted by the steroid hormone inhibitory effect of the MCH system during lactation. This effect is also reflected in the MCH serum levels.

Identification and quantification of vitellogenin gene in eyebrow goby (Oxyurichthys ophthalmonema)

Abstract. Sa-An AC, Quinitio GF, Anasco NC, Traifalgar RFM, Nillos MGG. 2022. Identification and quantification of vitellogenin gene in eyebrow goby (Oxyurichthys ophthalmonema). Biodiversitas 23: 5606-5613. Vitellogenin (VTG) gene has been used as a biomarker for exposure to estrogenic compounds in marine and freshwater fish species. There was limited information about the detection of VTG mRNA in an estuarine environment using a bioindicator fish species. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was developed for quantification of VTG mRNA normalized to ?-actin in eyebrow goby, Oxyurichthys ophthalmonema. VTG mRNA in the liver of reproductively mature female samples was isolated, amplified, and sequenced. There were 430 bp fragments of VTG, corresponding to a 143 amino acid sequence obtained from eyebrow goby. The partial protein sequence exhibited similar identities with the phosvitin motif of VTG in other gobies. The new qPCR assay was developed through the evaluation of hepatic mRNA levels in nonreproductive male eyebrow goby at 0, 6, 12, and 24 h following intraperitoneal injection of 10 mg kg-1 17?-estradiol. Hepatic mRNA levels were detected 6 h after 17?-estradiol injection and the highest VTG expression was noted 24 h after injection. Results of the present study suggest that male eyebrow goby can be used as indicator species for estrogenic contamination. Likewise, the established qPCR assay can be used to assess recent exposure to estrogenic contaminants in the aquatic environment. The characterization of the amplified VTG gene from eyebrow goby and the application of the qPCR assay developed for eyebrow goby in monitoring EDCs in the field may be considered for future investigation.

Sex-dependent changes of hippocampal synaptic plasticity and cognitive performance in C57BL/6J mice exposed to neonatal repeated maternal separation

The repeated maternal separation (RMS) is a useful experimental model useful in rodents to study the long-term influence of early-life stress on brain neurophysiology. We here investigated the influence of RMS exposure on hippocampal inhibitory and excitatory synaptic transmission, long-term synaptic plasticity and the related potential alterations in learning and memory performance in adult male and female C57Bl/6J mice. Mice were separated daily from their dam for 360 min, from postnatal day 2 (PND2) to PND17, and experiments were performed at PND 60. Patch-clamp recordings in hippocampal CA1 pyramidal neurons revealed a significant enhancement of GABAergic miniature IPSC (mIPSC) frequency, and a decrease in the amplitude of glutamatergic mEPSCs in male mice exposed to RMS. Only a slight but significant reduction in the amplitude of GABAergic mIPSCs was observed in females exposed to RMS compared to the relative controls. A marked increase in long-term depression (LTD) at CA3-CA1 glutamatergic synapses and in the response to the CB1r agonist win55,212 were detected in RMS male, but not female mice. An impaired spatial memory and a reduced preference for novelty was observed in males exposed to RMS but not in females. A single injection of β-ethynyl estradiol at PND2, prevented the changes observed in RMS male mice, suggesting that estrogens may play a protective role early in life against the exposure to stressful conditions. Our findings strengthen the idea of a sex-dependent influence of RMS on long-lasting modifications in synaptic transmission, effects that may be relevant for cognitive performance.

Modulatory effects of 17β-estradiol on acute lung inflammation after total occlusion of the descending aorta in male rats

As a consequence of systemic inflammation caused by ischemia and reperfusion (I/R) due to aortic occlusion, the lungs can exhibit increased microvascular permeability, local release of pro-inflammatory mediators, and leukocyte infiltration. Lung tissue infiltration by activated neutrophils is followed by acute respiratory distress syndrome, which is linked to acute pulmonary microvascular damage, high mortality rates, and organ dysfunction. Previous studies have demonstrated that female sex hormones modulate the inflammatory response and that prophylactic treatment with 17β-estradiol (E2) can prevent fatalities and preserve mesenteric perfusion and intestinal integrity after ischemia/reperfusion induced by aortic occlusion. In this study, we focused on the protective effects of estradiol after aortic ischemia/reperfusion by evaluating lung injury and endothelial alterations. Upon anesthesia and mechanical ventilation, male rats were subjected to aortic occlusion for 20 min, followed by 2 h of reperfusion. In parallel, one group of rats received a single injection of estradiol (280 µg/kg, i.v.) 30 min before ischemia. We observed increased serum concentrations of IL-1β, IL-6 and IL-10 in the I/R rats and E2 was able to reduce them. E2 effects after 2 h of reperfusion resulted mainly in decreasing of edema, iNOS expression and preventing leukocyte infiltration. Overall, our data indicate that estradiol might be a supplementary approach to deal with systemic processes and lung deterioration.

Distinct Roles of Estrogen Receptors in the Regulation of Vitellogenin Expression in Orange-Spotted Grouper (Epinephelus coioides).

During their breeding season, estrogen induces vitellogenin (VTG) production in the liver of teleost fish through estrogen receptors (ERs) that support oocyte vitellogenesis. There are at least three ER subtypes in teleost fish, but their roles in mediating E2-induced VTG expression have yet to be ascertained. In this study, we investigated the expression of vtgs and ers in the liver of orange-spotted grouper (Epinephelus coioides). Their expression levels were significantly increased in the breeding season and were upregulated by an estradiol (E2) injection in female fish, except for the expression of erβ1. The upregulation of vtgs, erα and erβ2 by E2 was also observed in primary hepatocytes, but these stimulatory effects could be abolished by ER antagonist ICI182780 treatment. Subsequent studies showed that ERβ antagonist Cyclofenil downregulated the E2-induced expression of vtg, erα, and erβ2, while the ERβ agonist DPN simulated their expression. Knockdown of erβ2 by siRNA further confirmed that ERβ2 mediated the E2-induced expression of vtgs and erα. To reveal the mechanism of ERβ2 in the regulation of erα expression, the erα promoter was cloned, and its activity was examined in cells. E2 treatment simulated the activity of the erα promoter in the presence of ERβ2. Deletions and site-directed mutations showed that the E2 up-regulated transcriptional activity of erα occurs through a classical half-estrogen response element- (ERE) dependent pathway. This study reveals the roles of ER subtypes in VTG expression in orange-spotted grouper and provides a possible explanation for the rapid and efficient VTG production in this species during the breeding season.

Estradiol administration in Holstein heifer calves differentially affects the fatty acid composition of subcutaneous adipose and the mammary fat pad tissues.

This research communication reports the relative abundance of fatty acids in mammary fat pad (MFP) and subcutaneous adipose (SCA) tissues for Holstein heifer calves receiving 0, 3, or 12 daily injections of estradiol immediately prior to tissue collection. The objective of this study was to determine if the MFP and SCA fatty acid profiles were affected by estradiol administration and if such a response differs between adipose tissue depots. Twelve Holstein heifer calves were reared on a common diet and administered 12 daily injections prior to euthanasia. Injections were either daily injections of corn oil (n = 4; CON), 9 injections of corn oil followed by 3 injections of estradiol (n = 4; SHORT), or 12 injections of estradiol (n = 4; LONG). Fatty acids were extracted from collected MFP and SCA tissues samples and analyzed using gas chromatography. The MFP tissues contained a greater abundance of saturated fatty acids than SCA tissues which complemented a reduced abundance of mono-unsaturated fatty acids in the MFP than SCA. Extended duration of estradiol administration increased the abundance of total omega 3 fatty acids in both MFP and SCA tissues. There was a treatment by tissue interaction for several of the C18:1 and C18:2 isomers indicating that estradiol's effects on fatty acid uptake and metabolism are tissue specific. Additionally, C18 uptake and metabolisms may have important roles in mammary growth and development. Together, these results indicate that the MFP responds differently to estradiol administration than SCA tissues and that these alterations are associated with different degrees of induced mammary growth via estradiol.